Drug toxicity in humans has been difficult to predict and thus has elicited an expensive toll on pharmaceutical research productivity. Unexpected toxicities account for 30% of drug failures in clinical studies and most post marketing drug withdrawals (Kola and Landis (2004) Nat Rev Drug Discov. 3:711-5). Predicting drug side effects has been complicated by the fact that drugs have the potential to subvert normal cellular and physiological processes by a variety of mechanisms.
Toxic agents, including chemical warfare (CW) threat agents, biowarfare agents, as well as pharmaceutical substances can subvert normal cellular processes by a variety of mechanisms. However, despite the diversity of molecular mechanisms and the potential for development of new threat agents with novel mechanisms, common signaling pathways and regulatory processes can be identified. Toxicities can result from specific on- or off-target activities that result in cell death or apoptosis (e.g. inhibition of the anti-apoptotic molecule XIAP); mitochondrial dysfunction (e.g. blocking mitochondrial electron transport); endoplasmic reticulum stress (e.g. proteasome inhibition), or other cell damage; or by interfering with physiologically important feedback mechanisms (e.g. hERG channel). While assessment of some of these activities is standard practice in preclinical development, the assays and cell types employed for many of these are not relevant to the human disease setting and do not predict outcomes in man.
Drugs can also induce toxicity as a consequence of metabolic activation, and transformation into reactive electrophiles that can covalently bind with proteins or DNA, or deplete glutathione (Liebler and Guengerich (2005) Nat. Rev. Drug Discov. 4:410-420). Depending on the targets of these reactions and exposure variables, toxic drug effects can include tissue damage (e.g. liver or heart), allergic and immune reactions, as well as neoplasia. Unfortunately, due to the large number and complex regulation of drug metabolizing enzymes, the ability to predict metabolites and their associated toxicological risks has been limited (Stevens, (2006) Chem. Res. Toxicol. 19:1393-1401; Baillie, Chem. Res. Toxicol (2008) 21:129-137). Cell systems that provide an enhanced sampling of drug metabolizing enzymes may assist in the discovery of toxic metabolites.
Toxic agents, including cellular or metabolic poisons, as well as vesicants, may trigger apoptosis and necrosis pathways directly in exposed tissues (see Li et al. (2005) Toxicol Sci. 86:116-24; Rosenthal et al. (2001) J Invest Dermatol. 117:1566-73). Secondary effects on other cells and tissues also contribute to mortality and morbidity. These can involve inflammatory pathways that are triggered by the initial tissue damage, for example, from exposure to sulfur mustard. Inflammatory pathways can alter cell responses to stress and toxic stimuli, and can themselves be modulated by cell stress and metabolic pathways. (See Wellen et al. (2005) J Clin Invest. 115:1111-9; Cowan et al. (2003) J Appl Toxicol. 23:177-86). Inflammatory and proteolysis pathways have been implicated in the toxicity of both nerve and blister agents.
An understanding of the interaction of inflammatory and cell death pathways is of high interest for the development of effective countermeasures, particularly with respect to chemical and biowarfare agents. Desirable countermeasures include those that are applicable to multiple agents within a class. Potential countermeasures include anti-inflammatory drugs, protease inhibitors, and inhibitors of poly ADP ribose polymerase-1 (PARP-1). Bifunctional compounds containing anti-cholinesterase functional groups coupled to non-steroidal anti-inflammatory drugs have been recently described as potential therapeutic agents for nerve and blister agents. Serine protease inhibitors have shown efficacy in prolonging survival in animals exposed to soman, an organophosphate nerve agent, and may have activity against sulfur mustard. Inhibitors of PARP-1, a DNA repair enzyme that has also been shown to regulate cell death in addition to catalyzing the synthesis of ADP-ribose polymers, may have utility in nerve-agent induced neuronal degeneration as well as sulfur mustard skin damage. A better understanding of the pathologic mechanisms of action of chemical threat agents will help identify additional therapeutic candidates, as well as new therapies with potential utility against novel threat agents.
The development of effective countermeasures against toxic agents is of great military and public interest. Preferably, countermeasures will include approaches that protect the targeted cellular pathways and are applicable to multiple agents. The development of countermeasures may require characterization of intra and inter cellular responses to diverse toxic agents, methods for characterizing the mechanism of action of diverse toxic agents, and a method for rapidly identifying appropriate countermeasures. The present invention addresses these issues.
In the case of pharmaceutical agents, the identification of compounds that are least likely to induce toxicity in patients is of great importance. The present invention, through its ability to identify compounds with secondary or untoward activities, provides a method for selecting agents with reduced toxicity potential.